Oxygen-independent real-time monitoring of distinct biphasic glutamate release using dialysis electrode in rat striatum during anoxia: In vivo evaluation of glutamate release and reversed uptake

Using a dialysis electrode, previous studies showed a clear biphasic releas e of glutamate during anoxia and ischemia. In this study, we examined two h ypotheses: (1) glutamate is of vesicular origin and its release is thus Ca2 +- and ATP-dependent in the first phase, while in the second phase glutamat e is derived primarily from the metabolic pool, and (2) reversed glutamate uptake, due to electrogenic stoichiometry, produces the second phase during anoxic insult in the rat brain. A dialysis electrode continuously perfused with glutamate oxidase and ferrocene-conjugated bovine serum albumin (BSA) optimized the time resolution of monitoring, allowing quantitative oxygen- independent, real-time measurement of the extracellular glutamate concentra tion ([Glu]e) during anoxia. [Glu]e dynamics were analyzed during anoxia by combining the dialysis electrode with focal microinjection of substances i nducing glutamate release. Following anoxia in the rat brain, a sharp and r apid [Glu]e elevation took place (first phase). The [Glu]e elevation then s hifted, continuing a gently sloping rise throughout the anoxic period (seco nd phase). This first phase disappeared with intracranial administration of either Co2+ or omega -conotoxin. The second phase rise increased with foca l microinjection of KCI (300 mM, 1 muL) and decreased with NaCl (300 mM, 1 CLL), ultimately reaching a plateau in both cases. Preloading with a novel glutamate transporter inhibitor (tPDC) decreased both the first and second phases of [Glu]e elevation. This dialysis electrode system provides data su pporting in vivo evidence that the peak of the first phase of [Glu]e elevat ion is derived from the "neurotransmitter pool," while the second phase is derived from the neuronal and glial "metabolic pool," which is, at least, p artly related to a "reversed uptake" mechanism in the anoxic rat brain.